Access Type

Open Access Dissertation

Date of Award

January 2015

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Tamara L. Hendrickson

Abstract

BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF THE CORE SUBUNITS OF GPI TRANSAMIDASE

by

DILANI G GAMAGE

May 2015

Advisor: Prof. Tamara L. Hendrickson

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

Glycosylphosphatidylinositol transamidase (GPI-T) is a complicated, membrane-bound, multi-subunit enzyme that catalyzes an essential post-translational modification. This enzyme attaches GPI anchors to the C-termini of various proteins that contain a proper GPI-T signal sequence. Gpi8, Gaa1, Gpi16, Gpi17 and Gab1 are the five known subunits that may encompass the fungal GPI-T; Gpi8 is the catalytic subunit, but the functions of the other subunits remain essentially unknown. In humans, different GPI-T subunits are upregulated in different cancers, making GPI-T a target for cancer research. However, in spite of the importance of this enzyme, little is known about how it assembles into an active enzyme complex, the stoichiometry of this complex, or the roles of the different components. Here we use soluble domains of the three core subunits (Gpi8, Gpi16 and Gaa1) to investigate the stoichiometry of the enzyme as well as to study the functions of each subunit in vitro. Additionally, overexpression of the full-length core subunits was used to study the enzyme's behavior on transamidation in vivo.

Due to the complex nature of this protein and the fact that it is membrane associated, we set out to simply this enzyme into a more tractable system. In chapters 2 and 3 of this thesis, we focused on the soluble domains of the core subunits, Gpi81-306, Gaa150-343 and Gpi161-551. These soluble domains were overexpressed and their interactions and stoichiometry were characterized. Gpi8, the catalytic subunit, has weak sequence similarity to caspase-1 and assembles into a homodimer. Also, N-linked glycosylation of one asparagine in this subunit is not essential for dimerization. Co-immunoprecipitation of the soluble domains of Gpi81-306:Gaa150-343, Gpi81-306:Gpi161-551 and Gpi161-551:Gaa150-343 demonstrated that these subunits interact with each other at least in heterodimeric complexes. Initial characterization of the Gpi823-306:Gaa150-343 complex is consistent with the formation of an  heterotetramer. Also, these three subunits Gpi81-306:Gaa150-343:Gpi161-551 can be co-purified as an intact complex. Preliminary results show that this core heterotrimer has nucleophile-independent activity. Our results will help to elucidate the function and resolve the complexity of GPI-T. Efforts are underway to determine the stoichiometry of each subunit and the contribution of each subunit towards transamidase activity.

To better understand how changes in expression affect GPI-T activity, and as a model for this enzyme in cancer, we have developed an in vivo strategy to monitor and quantify the effect of subunit overexpression on cell surface presentation of GPI-anchored proteins in Saccharomyces cerevisiae. Here we used Invertase as a reporter enzyme. Three GPI-T signal sequences were appended to the C-terminus of invertase and the amount of cell surfaced, GPI anchored invertase was measured. Overexpression of Gpi8, the catalytic subunit had little effect on GPI anchoring of invertase with two of these three signal sequences; however, the amount of cell surface invertase was nearly doubled when the weakest signal sequence was used. Compared to Gpi8, overexpression of either Gpi16 or Gaa1 downregulated GPI-T activity with all three signal sequences. To our knowledge, these results represent the first direct examination of the impact of subunit overexpression directly on GPI-T activity. Our results suggest that overexpression of a single GPI-T subunit either disrupts assembly of active GPI-T or frees these subunits to participate different cellular functions.

The results presented in this dissertation represent the beginning of a new era aimed at understanding GPI-T and provide new tools and approaches to achieve this important goal.

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